这项工作得到了国家卫生研究院（R01 DK077195至R.M.A、R01 DK104641至R.M.A和D.R.B）的赠款支持。我们感谢斯图亚特·奥金博士在血液学/肿瘤学系、波士顿儿童医院、儿科系、哈佛医学院和达纳-法伯癌症研究所提供的宝贵意见。我们还感谢凯尔·科斯塔、玛丽·塔利恩蒂和哈比巴拉·肖贾伊萨迪博士（儿科部杨石博士实验室、新生儿医学部、儿科部、新生儿医学部、波士顿儿童医院、哈佛医学院）在技术援助和有益讨论方面给予的支持。

<ol>
	<li>Grange, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+and+functional+analysis+of+human+tumor+immune+infiltration+after+mechanical+and+enzymatic+disaggregation.">Phenotypic characterization and functional analysis of human tumor immune infiltration after mechanical and enzymatic disaggregation.</a> Journal of Immunological Methods. 372 (1-2), 119-126 (2011).</li><li>van den Brink, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+sequencing+reveals+dissociation-induced+gene+expression+in+tissue+subpopulations.">Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations.</a> Nature Methods. 14 (10), 935-936 (2017).</li><li>Seth, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+discrete+modes+of+spinal+cord+injury+on+bladder+muscle+contractility.">The impact of discrete modes of spinal cord injury on bladder muscle contractility.</a> BMC Urology. 13, 24 (2013).</li><li>Doyle, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inosine+attenuates+spontaneous+activity+in+the+rat+neurogenic+bladder+through+an+A2B+pathway.">Inosine attenuates spontaneous activity in the rat neurogenic bladder through an A2B pathway.</a> Scientific Reports. 7, 44416 (2017).</li><li>Gheinani, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+miRNA-regulated+networks,+hubs+of+signaling,+and+biomarkers+in+obstruction-induced+bladder+dysfunction.">Characterization of miRNA-regulated networks, hubs of signaling, and biomarkers in obstruction-induced bladder dysfunction.</a> JCI Insight. 2 (2), 89560 (2017).</li><li>Gheinani, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concordant+miRNA+and+mRNA+expression+profiles+in+humans+and+mice+with+bladder+outlet+obstruction.">Concordant miRNA and mRNA expression profiles in humans and mice with bladder outlet obstruction.</a> American Journal of Clinical and Experimental Urology. 6 (6), 219-233 (2018).</li><li>Krishna, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+contusion+model+of+severe+spinal+cord+injury+in+rats.">A contusion model of severe spinal cord injury in rats.</a> Journal of Visualized Experiments. (78), e50111 (2013).</li><li>The Tabula Muris Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+of+20+mouse+organs+creates+a+Tabula+Muris.">Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris.</a> Nature. 562 (7727), 367-372 (2018).</li><li>Gray, C. J., Boukouvalas, J., Szawelski, R. J., Wharton, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzyloxycarbonylphenylalanylcitrulline+p-nitroanilide+as+a+substrate+for+papain+and+other+plant+cysteine+proteinases.">Benzyloxycarbonylphenylalanylcitrulline p-nitroanilide as a substrate for papain and other plant cysteine proteinases.</a> Biochemical Journal. 219 (1), 325-328 (1984).</li><li>Feodorova, Y., Koch, M., Bultman, S., Michalakis, S., Solovei, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick+and+reliable+method+for+retina+dissociation+and+separation+of+rod+photoreceptor+perikarya+from+adult+mice.">Quick and reliable method for retina dissociation and separation of rod photoreceptor perikarya from adult mice.</a> MethodsX. 2, 39-46 (2015).</li><li>Aitken, K. J., Bagli, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bladder+extracellular+matrix.+Part+I:+architecture,+development+and+disease.">The bladder extracellular matrix. Part I: architecture, development and disease.</a> Nature Reviews Urology. 6 (11), 596-611 (2009).</li><li>Aitken, K. J., Bagli, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bladder+extracellular+matrix.+Part+II:+regenerative+applications.">The bladder extracellular matrix. Part II: regenerative applications.</a> Nature Reviews Urology. 6 (11), 612-621 (2009).</li><li>The ENCODE Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Yue, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+encyclopedia+of+DNA+elements+in+the+mouse+genome.">A comparative encyclopedia of DNA elements in the mouse genome.</a> Nature. 515 (7527), 355-364 (2014).</li><li>Wu, Y. E., Pan, L., Zuo, Y., Li, X., Hong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Activated+Cell+Populations+Using+Single-Cell+RNA-Seq.">Detecting Activated Cell Populations Using Single-Cell RNA-Seq.</a> Neuron. 96 (2), 313-329 (2017).</li></ol>

没有宣布任何利益冲突。

此处描述的鼠脊髓损伤模型提供了一种可重复的方法，由于膀胱收缩和外部尿道括约肌松弛之间失去协调，可产生功能性膀胱出口阻塞。这反过来又唤起了膀胱壁的深刻改造，早在受伤后2周，其特点是扩大尿道和光滑的肌肉隔间。在啮齿动物中实施SCI模型的关键步骤包括:（i） 在脊柱休克期间严格注意手动膀胱表达，在受伤后10-u201214天;（二） 营养丰富，以尽量减少体重减轻:（三） 缓解尿液烫伤的可能性，特别是用于超越反射失效返回的实验。该模型的局限性包括:在瞬时血尿期间，小鼠可能从血栓中排出尿道，另外，在手术后逆行射精后，来自精液库古鲁姆的雄性小鼠中。
此处描述的组织分离方法说明了考虑实验侮辱引起的组织结构变化的重要性，在这种情况下，SCI 之后的重大组织重塑可能会影响下游分析。随着单细胞分析的增加，必须确保在基因表达中观察到的差异不仅仅是分离引起的扰动的结果，而是真正代表与疾病模型相关的潜在生物变化。使用公开的表达数据使我们能够修改消化缓冲器的配方，以确保细胞外矩阵的有效消化，同时最大限度地提高可行性。在未来的应用中可以考虑的其他修改包括添加actinomycin D，以阻止对分离协议15敏感的即时早期基因的转录。
在分离组织或转移已经处于悬浮状态的细胞时，管道技术至关重要。为了减少剪切力对细胞的物理损害，在细胞重新悬念期间轻轻和缓慢地使用移液器非常重要。一般建议使用宽孔移液器提示。如果使用标准提示，则轻轻使用移液器细胞悬架，以避免剪切力，否则会损害细胞，这一点尤为重要。然而，使用细胞过滤器是不可避免的，但是，细胞浓度可以下降20%或更多，伴随着100μL或以上的体积损失。我们建议在紧张后确定细胞浓度，以确保精确的细胞计数。
在流细胞学中，FMO 控制提供背景的测量，因为信号从重叠的发射峰值出血。它们不是非特异性抗体结合的量度，也不是当抗体包含在该通道中时可能存在的背景染色。要考虑非特异性抗体结合，必须包括适当的同型控制:对于背景染色，需要包括负控制。综合起来，这些控制确保准确测量细胞群。

正常尿囊功能的扰动会导致许多人生活质量下降。为了更好地了解损伤或疾病如何破坏膀胱的正常功能，重要的是要探索膀胱内细胞的正常生物状态，以及它们在实验扰动下是如何变化的。然而，迄今为止，尿囊内的特定细胞群以及它们如何随损伤而变化，其特征尚未完全确定。
单细胞剖析方法，如流细胞切除术或单细胞RNA测序（scRNA-seq），有可能揭示膀胱内的特定细胞类型。然而，要使这些方法成为信息性组织，必须以不影响收获组织的生存能力、基因表达和代表性细胞群百分比的方式进行消化。采用酶分聚的方案可以通过不分青红皂白的蛋白酶活性1影响表面标记表达，从而通过流细胞学影响细胞识别，而分离过程本身可导致立即早期基因的诱导，正如范登布林克及其同事最近描述的那样。作者表明，虽然受分离影响的亚人口很少，但由于即时早期基因的表达水平较高，它可能会在批量表达研究中触发强烈的污染信号。此外，分离协议的持续时间影响了某些亚种群所特有的基因的体积表达水平。因此，在不考虑分离协议影响的情况下生成的单个细胞数据集可能会产生分离方法产生的基因表达变化，而不是基础生物学。这些观察表明，发表的单细胞转录经济学数据应谨慎解释，结果应通过独立方法进行验证。
虽然，苛刻和冗长的分离方法可能会改变细胞2中的基因表达:有效分离细胞对于准确表示存在的细胞类型至关重要。由于膀胱是一个复杂的器官，由多种细胞类型组成，因此一些群体，如尿道细胞或频闪细胞，可能相对代表不足，而其他细胞类型，如成纤维细胞存在于细胞外基质内，可能难以分离。分离变得更加具有挑战性，如果膀胱经历了重大的改造和纤维化，如在脊髓损伤3，4或膀胱出口障碍5，6观察到。
在这里，我们描述了脊髓损伤小鼠膀胱下游单细胞分析的优化组织分离方法。使用流细胞学，我们比较了四种酶消化方案，它们能够产生单个细胞悬浮，支持细胞存活率，并保持细胞群的正确比例。基于这一分析，我们得出结论，尽量减少细胞死亡、细胞聚集物、非细胞核酸和下游分析的潜在抑制剂对于实现高质量的数据至关重要。

<table>
	<tbody>
		<tr>
			<td>2.5 X Magnifying Loupes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7-0 Vicryl suture, 6.5mm needle 3/8 circle</td>
			<td>ETHICON</td>
			<td>J546</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m Cell Strainer</td>
			<td>Thermofisher</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase in BPBS, 0.5mM EDTA</td>
			<td>Millipore</td>
			<td>SCR005</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Filter Wide Orifice Pipettor Tips (1000 &#181;L)</td>
			<td>VWR</td>
			<td>89049-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Filter Wide Orifice Pipettor Tips (1000 &#181;L)</td>
			<td>VWR</td>
			<td>89049-168</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD326 (Ep-CAM), rat monoclonal, IgG2a, &#954;, affinity purified</td>
			<td>BioLegend</td>
			<td>118213</td>
			<td></td>
		</tr>
		<tr>
			<td>BB515 Rat Anti-Mouse CD45, rat monoclonal, IgG2b, &#954;, Clone 30-F11</td>
			<td>BD Biosciences</td>
			<td>564590</td>
			<td></td>
		</tr>
		<tr>
			<td>BONN Micro Dissecting Forceps, Straight, 1x2 teeth, 3.75&#34; length, 0.3mm tip width, 0.12mm teeth</td>
			<td>ROBOZ Surgical Instrument Company, Inc.</td>
			<td>RS-5172</td>
			<td>ROBOZ Surgical Instrument Company, Inc., Gaithersburg MD</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A9647-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>2115-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>CASTROVIEJO Micro Suturing Needle Holder, Straight with lock, 5.75&#34; length</td>
			<td>ROBOZ Surgical Instrument Company, Inc.</td>
			<td>RS-6412</td>
			<td>ROBOZ Surgical Instrument Company, Inc., Gaithersburg MD</td>
		</tr>
		<tr>
			<td>Cell Counting Kit, 30 dual-chambered slides, 60 counts, with trypan blue</td>
			<td>Biorad</td>
			<td>1450003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Staining Buffer</td>
			<td>BioLegend</td>
			<td>420201</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum</td>
			<td>Sigma</td>
			<td>C0130-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type III</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS004182</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type 6</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS005319</td>
			<td></td>
		</tr>
		<tr>
			<td>Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 &#38; Propidium Iodide (PI)</td>
			<td>Thermofisher</td>
			<td>V13241</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma</td>
			<td>D4693-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma</td>
			<td>DN25-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Enrofloxacin (Baytril)</td>
			<td>Bayer Health Care LLC,</td>
			<td>NADA # 140-913 Approved by FDA. Lot No.: AH01CGP</td>
			<td>2.27% Injectable Solution</td>
		</tr>
		<tr>
			<td>Falcon 15 ml conical centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 ml conical centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse Ly-6A/E (Sca-1) Antibody, rat monoclonal, IgG2a, &#954;, affinity purified</td>
			<td>BioLegend</td>
			<td>122505</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase from sheep testes, Type II</td>
			<td>Sigma</td>
			<td>H2126</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (100 &#181;m)</td>
			<td>Miltenyi Biotec, Inc.</td>
			<td>130-110-917</td>
			<td></td>
		</tr>
		<tr>
			<td>McPHERSON-VANNAS, Micro Dissecting Spring Scissors, Straight, 4&#34; length, 0.15mm tip width</td>
			<td>ROBOZ Surgical Instrument Company, Inc.</td>
			<td>RS-5630</td>
			<td>ROBOZ Surgical Instrument Company, Inc., Gaithersburg MD</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Patterson Veterinary</td>
			<td>07-891-7959</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS003119</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy5 anti-mouse CD19 Antibody, rat monoclonal, IgG2a, &#954;, affinity purified</td>
			<td>BioLegend</td>
			<td>115509</td>
			<td>Dump Channel</td>
		</tr>
		<tr>
			<td>PE/Cy5 anti-mouse CD3&#949; Antibody, Armenian hamster monoclonal, IgG, affinity purified</td>
			<td>BioLegend</td>
			<td>100309</td>
			<td>Dump Channel</td>
		</tr>
		<tr>
			<td>PE/Cy5 anti-mouse CD4 Antibody, rat monoclonal, IgG2b, &#954;, affinity purified</td>
			<td>BioLegend</td>
			<td>100409</td>
			<td>Dump Channel</td>
		</tr>
		<tr>
			<td>PE/Cy5 anti-mouse CD8a Antibody, rat monoclonal, IgG2a, &#954;, affinity purified</td>
			<td>BioLegend</td>
			<td>100709</td>
			<td>Dump Channel</td>
		</tr>
		<tr>
			<td>PE/Cy5 anti-mouse NK-1.1 Antibody, mouse monoclonal, IgG2a, &#954;, affinity purified</td>
			<td>BioLegend</td>
			<td>108715</td>
			<td>Dump Channel</td>
		</tr>
		<tr>
			<td>PE/Cy5 anti-mouse TER-119/Erythroid Cells Antibody, IgG2b, &#954;, affinity purified</td>
			<td>BioLegend</td>
			<td>116209</td>
			<td>Dump Channel</td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block), rat monoclonal, IgG2b, &#954;, Clone 2.4G2</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC Lysis Buffer (10X)</td>
			<td>BioLegend</td>
			<td>420301</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer 1x</td>
			<td>Biolegend</td>
			<td>420201</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw-Cap microcentrifuge tubes, 1.5 ml</td>
			<td>VWR</td>
			<td>89004-290</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>Biorad</td>
			<td>1450102</td>
			<td></td>
		</tr>
		<tr>
			<td>Triple antibiotic ointment (neomycin/polymyxin B/ bacitracin)</td>
			<td>Patterson Veterinary</td>
			<td>07-893-7216</td>
			<td>skin protectant</td>
		</tr>
		<tr>
			<td>TrypLE Select Enzyme (10X), no phenol red</td>
			<td>Thermofisher</td>
			<td>A1217701</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetropolycin eye ointment</td>
			<td>Dechra Veterinary Products</td>
			<td>NADA # 065-016. Approved by FDA.</td>
			<td>protect eyes during anesthesia</td>
		</tr>
	</tbody>
</table>

a single cell dissociation approach for molecular analysis of urinary bladder in the mouse following spinal cord injury

我们描述了脊髓损伤在小鼠的实施，以引起脱毛器括约肌发育不良，功能性膀胱出口阻塞，以及随后的膀胱壁改造。为了促进评估非损伤控制和脊髓损伤小鼠膀胱壁的细胞组成，我们开发了一个优化的分离方案，支持高细胞存活率，并允许通过流细胞检测离散亚人口。
脊髓损伤是由胸脊髓的完全转位造成的。在组织收获时，动物在深度麻醉下注入磷酸盐缓冲盐水，膀胱被收获到Tyrode的缓冲液中。组织在消化缓冲区孵化之前切碎，消化缓冲区根据小鼠膀胱的胶原蛋白含量进行优化，通过对公开的基因表达数据库的询问确定。单细胞悬浮后，通过流细胞学分析材料，以评估细胞存活率、细胞数和特定子群落。我们证明，该方法产生细胞群超过90%的生存能力，并有力地表示细胞的间质和上皮起源。这种方法将能准确分析小鼠膀胱和其他器官中的离散细胞类型。

手术 胸脊髓转选的成功取决于对若干参数的评估，其中最明显的是后肢瘫痪。动物只用前肢移动，拖着后肢。否则，活动水平，包括喂养，梳妆和警觉性通常是正常的。此外，动物失去意志性膀胱控制，导致研究者每12小时需要手动膀胱表达，直到反射无效，受伤后10至14天返回。安乐死后，损伤成功的额外迹象主要与膀胱与体重比的增加有关，表明组织重塑。组理分析显示尿道和光滑的肌肉隔间3内增生。
准备单细胞悬架 利用公开的表达数据，确定细胞外基质蛋白的膀胱组织富集（图2），用于为消化组合的配方提供信息。由于胶原蛋白是膀胱壁11，12的关键组成部分，首先我们试图确定最丰富的胶原蛋白（s）在鼠标膀胱使用RNA分析数据集产生的鼠标ENCODE项目13。我们的分析表明，胶原蛋白1A1、胶原蛋白3A1、胶原蛋白1A2和胶原蛋白6A1是小鼠膀胱内最丰富的胶原蛋白类型（图2A）。我们还使用 Tabula Muris（鼠标（肌肉）的单细胞转录体数据简编）8来确定胶原蛋白 1、3、6 和透明质的 mRNA 表达水平。这些数据允许直接和对组织之间共享的细胞类型的基因表达进行对比。分析表明，这些细胞外基质组件的表达在间质细胞类型中更为普遍，而不是尿素（图2B）。
分离对膀胱中分离细胞生存能力的影响 流细胞学分析表明，采用4种不同方案的酶消化的存活率分别为83%、86%、93%和90%。因此，协议第3节被认为对保持细胞存活性最有价值。我们还观察到，大约4%的细胞是坏死细胞（PI+/附件V-）（图4A）。这些观察强调了消化方案的效率以及随后对细胞生存能力的好处。
脊髓损伤对膀胱中不同细胞群的影响 与对照组相比，我们观察到SCI小鼠膀胱的总细胞数量显著增加。从SCI膀胱获得的点图模式也略有不同，与脊髓损伤引起的器官重塑（图4B:第一列）一致。与对照组相比，SCI动物的膀胱显示CD45阳性细胞显著增加。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61455/61455fig01.jpg"> 图1:代表输液完成与肝脏的浅色。（A） 在输液开始时显示肝脏颜色。（B） 在输液结束时显示减轻的肝脏颜色。（A）中的小鼠在输液前两周进行脊髓转选，导致膀胱肥大，其突出出骨盆不像（B）中没有脊髓损伤的鼠标:在这种情况下，膀胱很小，隐藏在骨盆里。 <a href="https://www.jove.com/files/ftp_upload/61455/61455fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61455/61455fig02.jpg"> 图2:小鼠膀胱细胞外基质（ECM）组件的转录表达。（A） 43种不同胶原蛋白类型的条形图。该表达方式由每千万个映射读数（RPKM）（数据来自生物项目:PRJNA66167）14表示。（B） 在一池男性和女性分离的尿囊样本（男性和女性）中，从基于微流体液滴的3'end计数中获得的细胞类型的小提琴基因表达图谱。使用每百万 ln （CPM+1）8的自然对数，每个单元的计数均进行日志规范化。在进行对数之前添加了 1 个 CPM 的伪计数。<a href="https://www.jove.com/files/ftp_upload/61455/61455fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61455/61455fig03.jpg"> 图3:控制策略和FMO控制，以确定荧光扩散。（A） 细胞种群的选择。（B） 单打的格子策略。（C） 使用PI和附件V抗体对坏死细胞和早晚凋亡细胞进行治疗。（D_2012F）多色流细胞学的示意图点图（例如，与 A、B、C、D 荧光素 + （附件 V 和 PI）结合的抗体）。与未染色控制相比，FMO 控制显示的荧光通过荧光 A 通道扩散到抗体中。橙色虚线表示 FMO 界点边界，而未染色的边界为红色。 <a href="https://www.jove.com/files/ftp_upload/61455/61455fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61455/61455fig04.jpg"> 图4:膀胱中不同细胞类型的流细胞学。（A） 附件 V/PI 双染色流程图。每个协议所使用的酶和化学成分的不同组合在相应的生存图前表示。这些数据表明，通过协议第 3 节获得了最高的可行性。（B） 代表直方图，说明在单个通道中检测到的 Ly-6A/E （Sca-1） 和 CD326 （Ep-CAM） 的强度。（ C） SCI对小鼠膀胱细胞群的影响。上面板显示从对照非手术小鼠获得的三个分离膀胱上的染色结果，下面板显示三个带有SCI的动物染色的结果。第一栏是细胞总数。第二栏显示单项网格选择。第三栏显示了对B细胞、T细胞和NK细胞呈阴性的活细胞的亚群。第四栏显示活细胞的染色对CD45呈阳性。 <a href="https://www.jove.com/files/ftp_upload/61455/61455fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>金额（500 mL）</td>
			<td>摩尔 浓度</td>
		</tr>
<tr>
<td>Nacl</td>
			<td>4.091 克</td>
			<td>140米</td>
		</tr>
<tr>
<td>氯化钾</td>
			<td>0.186 克</td>
			<td>5米</td>
		</tr>
<tr>
<td>MgCl2
</td>
			<td>0.0476 克</td>
			<td>1米</td>
		</tr>
<tr>
<td>D-葡萄糖</td>
			<td>0.9 克</td>
			<td>10米</td>
		</tr>
<tr>
<td>海佩斯</td>
			<td>1.19 克</td>
			<td>10米</td>
		</tr>
</tbody></table>表1:泰罗德解决方案准备的组件。 指示的组件用于准备 500 mL 泰罗德的解决方案。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>量</td>
			<td>协议第 1 节</td>
			<td>协议第2节</td>
			<td>协议第3节</td>
			<td>协议第4节</td>
		</tr>
<tr>
<td>Bsa</td>
			<td>5毫克</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>卡克2
</td>
			<td>0.03 mM</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>拼贴酶 I 型</td>
			<td>132.5 单位</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>拼贴酶类型 I</td>
			<td>96.4 单位</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>拼贴酶类型六</td>
			<td>50 个单位</td>
			<td>-</td>
			<td>-</td>
			<td>是的</td>
			<td>-</td>
		</tr>
<tr>
<td>纳塞</td>
			<td>10 个单位</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
			<td>-</td>
		</tr>
<tr>
<td>木瓜 蛋白酶</td>
			<td>115 单位</td>
			<td>-</td>
			<td>-</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>潘拼贴画</td>
			<td>50 个单位</td>
			<td>-</td>
			<td>-</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>透明 质 酸 酶</td>
			<td>10.5 单位</td>
			<td>-</td>
			<td>-</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>迪斯帕塞二世</td>
			<td>1.25 单位</td>
			<td>是的</td>
			<td>是的</td>
			<td>-</td>
			<td>-</td>
		</tr>
<tr>
<td>细胞分离溶液</td>
			<td>1 mL</td>
			<td>是的</td>
			<td>-</td>
			<td>是的</td>
			<td>-</td>
		</tr>
<tr>
<td>重组酶</td>
			<td>1 mL</td>
			<td>-</td>
			<td>是的</td>
			<td>-</td>
			<td>是的</td>
		</tr>
</tbody></table>表2:用于准备消化缓冲液的组件。 所示组件用于制备 2.5 mL 消化混合物（1 U 催化 1 μmol 每分钟 37 °C 的基板水解。 有关每个酶单位的定义，请参阅产品数据表）。

Watch this Scientific Journal Video about A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury at JoVE.com

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

此协议的目标是将优化的组织分离协议应用于脊髓损伤的鼠标模型，并验证通过流细胞学进行单细胞分析的方法。

脊髓损伤后小鼠泌尿膀胱分子分析的单细胞分离方法

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and ...

a-single-cell-dissociation-approach-for-molecular-analysis-urinary

Research

JoVE Journal

Biology

5.4K 次观看.  Boston Children's Hospital. 此协议的目标是将优化的组织分离协议应用于脊髓损伤的鼠标模型，并验证通过流细胞学进行单细胞分析的方法。

视频: A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

TRE重组酶的分子进化

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

科研

生物学

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

压和外尿道括约肌测量在清醒大鼠植入导管和电极允许重复测量

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

单细胞微流控芯片分析枯草芽孢杆菌

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

绿松石鳉鱼实验室外壳的协议 (Nothobranchiusfurzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...